Mycobacterium tuberculosis is the causative agent of human tuberculosis. The bacterium has the capacity to persist in its human host for decades prior to progressing to active disease. In fact, on balance, humans deal with M. tuberculosis infection quite effectively with only an estimated 5 to 10% of those infected actually ending up with clinical disease. However, because of the extraordinary penetrance of this infectious agent across the global population, this accounts for in excess of 1 million deaths due to tuberculosis every year. The combination with HIV in sub-Saharan Africa is catastrophic, and M. tuberculosis is now the leading cause of mortality among individuals living with HIV.

Life and death dynamics during long-term intracellular survival of M. tuberculosis. (A) Survival assays. Resting murine bone marrow-derived macrophages were infected at low multiplicity of infection (∼1:1) with M. tuberculosis CDC1551. Viable CFU were quantified at day 0 and at 2-day intervals postinfection (p.i.) over a 14-day time course by lysis of monolayers, serial dilution, and plating on 7H10 medium. Error bars indicate standard error of the mean. (B) Replication clock plasmid. The percentage of bacteria containing the pBP10 plasmid during growth in resting macrophages was determined by comparing CFU (mean ± standard deviation) recovered on kanamycin versus nonselective media (red). The cumulative bacterial burden (CBB) (black) was determined by mathematical modeling based on total viable CFU and plasmid frequency data. Data shown represent two independent experiments. (C) The “bottleneck” response. Temporal expression profiles of genes differentially regulated at day 2 p.i., including genes from A that were upregulated (red) or downregulated (blue) >1.5-fold (shown as ratio of signal intensity relative to control). Note the maximal change in transcript levels at day 2 p.i. followed by majority trending back toward control levels. (D) “Guilt by association” analysis. Genes regulated in sync with known virulence regulons. i.e., the DosR regulon, were identified by using a highly regulated member of this regulon, hspX, in place of synthetic profiles. This figure is reproduced from Rohde et al. (24).

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Figure 1

Life and death dynamics during long-term intracellular survival of M. tuberculosis. (A) Survival assays. Resting murine bone marrow-derived macrophages were infected at low multiplicity of infection (∼1:1) with M. tuberculosis CDC1551. Viable CFU were quantified at day 0 and at 2-day intervals postinfection (p.i.) over a 14-day time course by lysis of monolayers, serial dilution, and plating on 7H10 medium. Error bars indicate standard error of the mean. (B) Replication clock plasmid. The percentage of bacteria containing the pBP10 plasmid during growth in resting macrophages was determined by comparing CFU (mean ± standard deviation) recovered on kanamycin versus nonselective media (red). The cumulative bacterial burden (CBB) (black) was determined by mathematical modeling based on total viable CFU and plasmid frequency data. Data shown represent two independent experiments. (C) The “bottleneck” response. Temporal expression profiles of genes differentially regulated at day 2 p.i., including genes from A that were upregulated (red) or downregulated (blue) >1.5-fold (shown as ratio of signal intensity relative to control). Note the maximal change in transcript levels at day 2 p.i. followed by majority trending back toward control levels. (D) “Guilt by association” analysis. Genes regulated in sync with known virulence regulons. i.e., the DosR regulon, were identified by using a highly regulated member of this regulon, hspX, in place of synthetic profiles. This figure is reproduced from Rohde et al. (24).

Demonstrating the usefulness of the hspX′::GFP reporter strain in assessing and reporting on the localized induction of iNOS at the site of infection. Phosphate-buffered saline-immunized (naive) mice and mice vaccinated with heat-killed M. tuberculosis (vac) were infected with the hspX′::GFP, smyc′::mCherry Erdman M. tuberculosis reporter strain. Fluorescence induction of the hspX promoter-dependent GFP is higher at 14 days in the vaccinated animals, as assessed by confocal microscopy of thick tissue sections (A) that were scored subsequently by Volocity (B). (C) The thick tissue sections were probed with antibodies against murine NOS2 (magenta), demonstrating the colocalization between GFP induction and NOS2 expression at the site(s) of infection. N.S., not significant. Data shown are detailed in Sukumar et al. (31).

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Figure 2

Demonstrating the usefulness of the hspX′::GFP reporter strain in assessing and reporting on the localized induction of iNOS at the site of infection. Phosphate-buffered saline-immunized (naive) mice and mice vaccinated with heat-killed M. tuberculosis (vac) were infected with the hspX′::GFP, smyc′::mCherry Erdman M. tuberculosis reporter strain. Fluorescence induction of the hspX promoter-dependent GFP is higher at 14 days in the vaccinated animals, as assessed by confocal microscopy of thick tissue sections (A) that were scored subsequently by Volocity (B). (C) The thick tissue sections were probed with antibodies against murine NOS2 (magenta), demonstrating the colocalization between GFP induction and NOS2 expression at the site(s) of infection. N.S., not significant. Data shown are detailed in Sukumar et al. (31).

Flow sorting of activated and resting M. tuberculosis-infected host cells demonstrates that the drug sensitivity of M. tuberculosis recovered from in vivo infection correlates inversely with the immune activation status of the host phagocyte. (A to C)M. tuberculosis recovered from activated host cells in vivo were more tolerant to both INH and RIF than those recovered from resting host cells. C57BL/6J mice were infected with mCherry-expressing M. tuberculosis Erdman for 21 days, and M. tuberculosis-containing myeloid cells with different immune activation status isolated from lung tissue by using flow cytometry. (A) CD11b+ mCherry+ CD80high cells (activated population) and CD11b+ mCherry+ CD80low cells (resting population) were sorted according to the depicted gating strategies. (B and C) Isolated cells were established in culture and subjected to treatment with 1 μg/ml INH or RIF or an equivalent volume of dimethyl sulfoxide (DMSO). Following 24 h of drug treatment, bacterial survival was determined by CFU enumeration (B), and the percentage of M. tuberculosis surviving drug treatment was quantified by normalizing bacterial load in drug-treated samples against that in DMSO-treated samples (C). This figure is modified from the work of Liu et al. (34).

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Figure 3

Flow sorting of activated and resting M. tuberculosis-infected host cells demonstrates that the drug sensitivity of M. tuberculosis recovered from in vivo infection correlates inversely with the immune activation status of the host phagocyte. (A to C)M. tuberculosis recovered from activated host cells in vivo were more tolerant to both INH and RIF than those recovered from resting host cells. C57BL/6J mice were infected with mCherry-expressing M. tuberculosis Erdman for 21 days, and M. tuberculosis-containing myeloid cells with different immune activation status isolated from lung tissue by using flow cytometry. (A) CD11b+ mCherry+ CD80high cells (activated population) and CD11b+ mCherry+ CD80low cells (resting population) were sorted according to the depicted gating strategies. (B and C) Isolated cells were established in culture and subjected to treatment with 1 μg/ml INH or RIF or an equivalent volume of dimethyl sulfoxide (DMSO). Following 24 h of drug treatment, bacterial survival was determined by CFU enumeration (B), and the percentage of M. tuberculosis surviving drug treatment was quantified by normalizing bacterial load in drug-treated samples against that in DMSO-treated samples (C). This figure is modified from the work of Liu et al. (34).

Examination of the bacterial stress reporter M. tuberculosis strain hspX′::GFP, smyc′::mCherry Erdman at the level of different host phagocyte populations in murine lung infection model. (A) The flow cytometry gating strategy for the identification of M. tuberculosis-infected phagocyte subsets from infected mouse lung, showing preliminary identification of alveolar macrophages, recruited interstitial macrophages, and monocyte-derived dendritic cell populations. (B) The level of expression of NO-driven GFP under regulation of the hspX promoter, identifying those phagocytes that induce the highest level of bacterial stress, and how the stress intensifies from 14 to 28 days postinfection. The levels of induction of expression of GFP indicate that the most stressful host cells appear to be neutrophils and the least stressful appear to be alveolar macrophages. C. Labeling of the host cells with antibody against NOS2 demonstrates the direct correlation between expression levels of the host nitric oxide synthase, NOS2, and levels of expression of the bacterial stress response reporter hspX′::GFP, shown in panel B. MFI, mean fluorescence intensity; PMN, polymorphonuclear leukocytes; IM, recruited interstitial macrophages; AM, alveolar macrophages.

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Figure 4

Examination of the bacterial stress reporter M. tuberculosis strain hspX′::GFP, smyc′::mCherry Erdman at the level of different host phagocyte populations in murine lung infection model. (A) The flow cytometry gating strategy for the identification of M. tuberculosis-infected phagocyte subsets from infected mouse lung, showing preliminary identification of alveolar macrophages, recruited interstitial macrophages, and monocyte-derived dendritic cell populations. (B) The level of expression of NO-driven GFP under regulation of the hspX promoter, identifying those phagocytes that induce the highest level of bacterial stress, and how the stress intensifies from 14 to 28 days postinfection. The levels of induction of expression of GFP indicate that the most stressful host cells appear to be neutrophils and the least stressful appear to be alveolar macrophages. C. Labeling of the host cells with antibody against NOS2 demonstrates the direct correlation between expression levels of the host nitric oxide synthase, NOS2, and levels of expression of the bacterial stress response reporter hspX′::GFP, shown in panel B. MFI, mean fluorescence intensity; PMN, polymorphonuclear leukocytes; IM, recruited interstitial macrophages; AM, alveolar macrophages.

The prpD′::GFP reporter strain responds to cholesterol during infection. (A) GFP expression is induced with propionate and cholesterol. The GFP mean fluorescence intensity (MFI) was determined from 10,000 bacteria by flow cytometery. (B) Resting bone marrow-derived macrophages were infected with the M. tuberculosis prpD′::GFP reporter strain for 24 h. Within macrophages the prpD′::GFP reporter is active. (C) Treating macrophages infected with the M. tuberculosis prpD′::GFP reporter strain with one of the inhibitors of cholesterol breakdown, identified in a recent drug screen (36), abolishes induction of the reporter signal. Nuclei are stained with DAPI, and the images are courtesy of Kaley Wilburn. DAPI, 4′,6-diamidino-2-phenylindole.

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Figure 5

The prpD′::GFP reporter strain responds to cholesterol during infection. (A) GFP expression is induced with propionate and cholesterol. The GFP mean fluorescence intensity (MFI) was determined from 10,000 bacteria by flow cytometery. (B) Resting bone marrow-derived macrophages were infected with the M. tuberculosis prpD′::GFP reporter strain for 24 h. Within macrophages the prpD′::GFP reporter is active. (C) Treating macrophages infected with the M. tuberculosis prpD′::GFP reporter strain with one of the inhibitors of cholesterol breakdown, identified in a recent drug screen (36), abolishes induction of the reporter signal. Nuclei are stained with DAPI, and the images are courtesy of Kaley Wilburn. DAPI, 4′,6-diamidino-2-phenylindole.

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